NADPH oxidases (Nox) are proteins present in the membrane of a variety of mammalian cell types whose function is to produce reactive oxygen species (ROS) such as superoxide anion (O2−) and hydrogen peroxide (H2O2). Maintaining healthy O2− levels is important in maintaining vascular tone. However, excessive O2− production may lead to hypertension through scavenging of the vasodilatory agent nitric oxide (NO) [1]. In addition, Nox-dependent ROS production is critical in a number of cellular signaling pathways and is involved in multiple cardiovascular diseases and colon cancer. [See 6, 7.]
With this wide gamut of functions for ROS, tight regulation of Nox by the cell is required. This is accomplished in part by generation of a number of different isoforms of Nox that are differentially regulated and produced in varying cell types. Among these isoforms, Nox1 and Nox2 bear significant homology. Each of these isoforms associates with the membrane protein p22phox and cytosolic organizing and activating subunits. Nox1 was originally discovered in colon carcinoma cells and has more recently been found to cause deleterious vascular constriction and contribute to angiogenic processes [2]. Nox2 is the prototypical enzyme of the family and is responsible for the respiratory burst found in phagocytes [2].
Both Nox1 and Nox2 produce O2− exclusively while Nox4 is thought to primarily produce H2O2 [2]. Nox1 and Nox2 derived O2− directly inactivates NO. Cells use cytosolic cofactors to regulate Nox-derived ROS production. Nox4 appears not to require classical cytosolic factors as do Nox1 and 2 but recently Poldip2 has been described as modulatory for Nox4. Nox1 activation is regulated primarily by the cytosolic proteins NOXA1, NOXO1, and the GTPase Rac1 while Nox2 activation is primarily regulated by the cytosolic proteins p67phox, p47phox, p40phox, and the GTPase Rac2 [2].
NOXA1 shares 38.3% homology with p67phox and a specific domain of p67phox from amino acids 190-200 is particularly well conserved across the animal kingdom and shares high homology with NOXA1 [3-5] (FIG. 1A-B). This domain has been termed the activation domain of p67phox and within it are a number of amino acids critical to the activation of Nox2. A portion of NOXA1 stretching from amino acids 191-211 is highly homologous to the activation domain of p67phox. Within these amino acids, 199-201 are of particular importance to enzyme activation (FIG. 1A). If one of these amino acids in NOXA1 is mutated to an alanine, the protein's function is reduced and in some cases totally lost [5].